The invention relates to a method for producing a nuclease of a Gram-negative bacterium or a nuclease preparation containing a nuclease of a Gram-negative bacterium comprising the expression of the nuclease in a Gram-positive bacterium and the subsequent secretion of the nuclease.
Nucleases are hydrolytic enzymes that split nucleic acids and are of widespread economic importance. Specific nucleases such as restriction enzymes are distinguished here from non-specific nucleases such as RNase A. Restriction enzymes have become indispensable tools in molecular biology and serve to specifically split different DNA molecules, which are joined together using ligases to form new constructs. Non-specific nucleases are mainly used for the decomposition of nucleic acids in various processes. When the nuclease cleaves only DNA, this is referred to as a “DNase”, and when the nuclease cleaves only RNA, this is referred to as an “RNase”. A typical representative of DNases is DNase I from the pancreas of mammals. Typical representatives of RNases are, for example, RNase T1 and T2 from Aspergillus oryzae or RNase A also from the pancreas of mammals.
Besides the application for removing RNA from DNA samples by treating with RNases or removing DNA from RNA samples by treating with DNases, further applications of significant economic interest are those in which both DNA and RNA are removed from the sample. This relates, for example, to the production of a wide variety of molecules by cell-based or cell-free biological systems, in which the product is not composed of nucleic acids, as in the case of proteins such as antibodies or enzymes, polysaccharides, lipids, for example, or low-molecular substances such as antibiotics, metabolic end products or intermediate products or chemicals. The necessity to remove the nucleic acids becomes particularly significant if production of the molecules occurs intracellularly or if a proportion of the production cells is lysed during production. As a result of this, during preparation of the molecules large amounts of nucleic acids are also released or are contained in the preparation which contaminate the desired molecule or make further purification thereof more difficult. A similar problem results, for example, in the production of proteins using cell-free in-vitro translation. The purification is made difficult, amongst other factors, by the nucleic acids increasing the viscosity of the preparations to such an extent that subsequent steps such as filtration or chromatography operations are not possible.
Hence, there is great interest in such processes to remove the contaminating nucleic acids or to digest these to such an extent that no further restriction to the further process steps occurs. One possibility of removing the nucleic acids consists in the specific precipitation of the nucleic acids by different agents. Another possibility consists in breaking down the nucleic acids to such small fragments using nucleases that the viscosity of the samples is reduced and the resulting decomposition products can be separated using simple methods such as e.g. ultrafiltration.
The use of nucleases that can cleave both RNA and DNA is particularly advantageous for an application for removal of all nucleic acids, i.e. both RNA and DNA, from different samples. In this case, the nuclease used should have a high activity and sufficient stability. A nuclease that exhibits these properties is the nuclease from the Gram-negative bacterium Serratia marcescens [EC 3.1.30.2; SEQ ID 1, Filimonova M N, Balaban N P, Sharipova F P, Leshchinskaia I B, Biokhimiia, 1980, 45(11): 2096-104; Filimonova M N, Baratova L A, Vospel'nikova N D, Zheltova A O, Leshchinskaia I B, Biokhimiia, 1981, 46(9): 1660-6; Ball T K, Saurugger P N, Benedik M J, Gene. 1987, 57(2-3): 183-92; Biedermann K, Jepsen P K, Riise E, Svendsen I, Carlsberg Res Commun. 1989, 54(1): 17-27]. This enzyme is also distributed under the brand name Benzonase and is referred to below as “Serratia marcescens nuclease”.
To be able to produce proteins economically in sufficient quantities and with the required purity, they are frequently produced using standard expression organisms by heterologous expression, i.e. the genetic information for the desired protein is incorporated into the expression organism, which then undertakes the expression, i.e. synthesis, of the protein foreign to it. This frequently has the advantage that the yield in these expression organisms can be increased very significantly compared to the original organism and established processes are available for cultivation of the expression organisms and their further treatment for product fabrication.
Nucleases can exert a high toxic potential on the host organism in the case of a disturbed or defective expression. If the nuclease already changes into an active form in the cytosol, it would split the nucleic acids of the host and cause them to die or inhibit their growth. Equally, a fault in folding or secretion can cause the secretion mechanism of the host to be blocked or impaired, which can also lead to death or growth inhibition.
The recombinant expression of the Serratia marcescens nuclease in the Gram-negative bacterium Escherichia coli is described in patent EP 229 866 B1 and described in comparison to the expression yield in the wild strain—Serratia marcescens W225. It is shown in Table 3 in page 13 and page 14 that with the system used a nuclease yield of 35 units/ml of culture was obtained with the recombinant E. coli strain and 7 units/ml with the wild strain. Moreover, it is disclosed that approximately half the activity remains in the periplasm of E. coli and is not secreted into the medium (Table 4 in EP 229 866 B1).
Biedermann K, Fiedler H, Larsen B S, Riise E, Emborg C, Jepsen P K, Appl. Environ. Microbiol. 1990, 56(6): 1833-8 also describe the secretion of a Serratia marcescens nuclease (from the Serratia marcescens strain W280) in E. coli. The study shows a comparison of the secretion rates of the nuclease in the homologous Gram-negative host organism Serratia marcescens and the likewise Gram-negative model organism E. coli. Nuclease yields per ml of culture under fermentation conditions that correspond to 16 500 units/ml are reported in the publication (Table 1, page 1837).
There are also papers in the prior art that relate to the homologous expression of a ribonuclease in the Gram-positive host Bacillus subtilis (Nakamura A, Koide Y, Miyazaki H, Kitamura A, Masaki H, Beppu T, Uozumi T, Eur. J. Biochem. 1992, 209(1): 121-127). Secretion yields of 7.2 units/ml are reported.
An expression of a heterologous nuclease in a Gram-positive bacterium is also described in the prior art (Dieye Y, Usai S, Clier A, Gruss A, Piard J-C, J. Bact. 2001, 183(14): 4157-4166). In this study the nuclease from the Gram-positive bacterium Staphylococcus aureus is expressed in the Gram-positive bacterium Lactobacillus lactis. The aim of this study is in particular to establish a system for the expression of desired proteins in the intestine of humans or animals by L. lactis (page 4157, left column).
The Gram staining is an important criterion for the differentiation of bacteria according to the structure of their cell wall. It is based on the different structure of the bacterial envelope composed of different peptidoglycans as well as teichoic acids. Gram-positive bacteria in this case have a thicker multilayer murein envelope that can represent up to almost 50% of the envelope dry mass. In addition, the cell wall contains between 20% and 40% teichoic acids. In contrast, Gram-negative bacteria have only a thin single-layer murein envelope, which only represents about 10% of the dry mass of the bacterial envelope and does not contain any teichoic acids. Methods for conducting the Gram staining are known to the person skilled in the art. Examples of Gram-negative bacteria are all types of the proteobacteria division such as enterobacteria (Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter) or Pseudomonas, Legionella, Neisseria, Serratia marcescens, the original host of the Serratio marcescens nuclease, is likewise a Gram-negative bacterium. Examples of Gram-positive bacteria are actinobacteria and strains of the Firmicutes (e.g. Streptococcus, Enterococcus, Staphylococcus, Listeria, Bacillus, Clostridium, Lactobacillus).
Gram-negative bacteria in general and E. coli in particular are distinguished by some disadvantages. On the one hand, secretion is often possible only in small yields and generally leads only into the periplasm and not directly into the medium, which makes possibly necessary subsequent purifications more difficult. On the other hand, Gram-negative bacteria often form endotoxins on a large scale. They are formed from a hydrophilic polysaccharide component and a lipophilic lipid component. In contrast to the bacteria they come from, endotoxins are highly heat-stable and even survive sterilisation. Endotoxins belong to the pyrogens, i.e. they can generate fever in humans and many types of animals upon contact with mucous membranes and passage into the bloodstream. Moreover, they activate a series of signalling pathways from immunocompetent cells that can either cause inflammation or a programmed cell death (apoptosis) of these cells. They are already biologically active in extremely low concentrations (lower pg/mL range).
Consequently, complex purification processes are necessary to reduce these endotoxins to below the biologically active concentration from samples, which can pass directly or indirectly into the human or animal bloodstream. This situation is particularly relevant for pharmaceutical applications.